Controlling cell-cell signaling at the microscale has led directly to advances in fundamental cell biology and re-generative medicine. Our goal is to develop technology to modulate cell signaling in vitro, specifically focusing on embryonic stem cells. While conventional techniques exist for probing intercellular communication, these methods provide incomplete control over the cell-culture microenvironment. A host of new technologies, such as cell patterning and microfluidic culture, have resulted in new ways to study cell- cell interactions. Despite the achievements of these technologies, limitations arise as we try to apply them to stem cell biology. In particular, cell-patterning techniques typically require patterning the substrate, which is a hindrance when studying proliferating stem cells. While microfluidic perfusion culture has been postulated to affect autocrine/paracrine signaling, this has not been rigorously demonstrated, and thus the full potential of microfluidic perfusion for modulating diffusible signaling has not been realized. We believe that addressing these limitations will enable new ways of studying not only stem cell signaling, but signal transduction in other cell systems as well. We are particularly interested in stem cells because they are powerful models for developmental and disease processes, as well as components for current and future therapeutics. To this end, we have developed two complementary technologies to study these cells: a new way to pattern cells and a microfluidic perfusion system. Our approach is to develop microtechnology that can (1) uses fluid flow in microfluidic perfusion chambers to modulate diffusible signaling, and (2) bio-flipchip cell patterning to place progenitor cells in defined arrangements, modulating diffusible and juxtacrine signaling. We will use these technologies to study mouse embryonic stem cell self-renewal and differentiation to neurons, and human embryonic stem cell self- renewal. Our specific aims are to (1) use microfluidic perfusion arrays to modulate mouse embryonic stem cell self-renewal and differentiation by controlling cell-cell diffusible signaling, (2) study direct and diffusible cell-cell interactions during passaging in mouse embryonic stem cell self-renewal using bio-flipchips, and (3) apply both technologies to studying human embryonic stem cell self-renewal.;;The relevance of this project to human health is in deciphering the mysteries of stem cell biology in order to use stem cells as therapeutics. Before stem cells can be used to treat disease, we must understand how to propogate them in culture and use them to create various tissues. Our technology provides a new window into stem cell biology that will help determine new ways to control their behavior.